Project Summary/Abstract: Alcohol use disorders (AUD) are a significant global health burden. AUDs are an established risk factor for bacterial pneumonia, which accounts for ~3.1 million deaths annually. AUD patients are more frequently infected with highly virulent respiratory pathogens and experience increased morbidity and mortality from these infections, with Klebsiella pneumoniae being overrepresented in patients with AUDs. Mortality from Klebsiella pneumonia in patients with AUDs is double that from other pathogens. Further, preclinical and clinical studies show that alcohol consumption perturbs the normal intestinal microbial communities (dysbiosis), yet no published data exist linking alcohol-mediated intestinal dysbiosis with respiratory host defense dysfunction and no attempt has been made to isolate the direct effects of alcohol from those resulting from intestinal dysbiosis. We have developed a mouse model system that enables us to investigate the immune modulatory effects of alcohol-associated dysbiosis and isolate the host responses to K. pneumoniae mediated directly or indirectly by ethanol-associated dysbiosis. Preliminary studies using fecal transfer show that alcohol-nave animals recolonized with microbiota isolated from alcohol-fed mice have increased susceptibility to K. pneumoniae compared to mice recolonized with a control microbiota. The overall hypothesis to be tested in the proposed study is that alcohol-mediated dysbiosis increases intestinal inflammation and permeability, which leads to intestinal sequestration of T-cells, altered lung specific T-cell trafficking, and increased susceptibility to Klebsiella pneumoniae. I will test my hypothesis using both a metabolomics and immunological approach. Aim 1 will test the prediction that alcohol-dysbiosis promotes intestinal inflammation and permeability. I will utilize fecal adoptive transfer and cell culture experiments to determine the microbial and metabolic constituents that promote intestinal inflammation and permeability. Aim 2 will examine the impact of alcohol- dysbiosis on intestinal T-cell programing and sequestration. I will use T-cell adoptive transfer experiments to determine the effects of alcohol-dysbiosis on the location/sequestration of T-cells prior to respiratory infection. Aim 3 will test the prediction that alcohol-dysbiosis impairs lung specific T-cell trafficking. I will assess T-cell trafficking using microbial and metabolite primed T-cells adoptively transferred into alcohol-nave animals recolonized with an alcohol-dysbiotic or pair-fed microbiota. The research and training plan proposed in this K99 application will provide me with additional training in alcohol- related immunology, molecular mechanisms involved in microbiota-mediated immune regulation, and metabolomics. The proposed studies will use a novel model system to clarify the role of the microbiota in host immune responses, particularly with regard to AUDs and respiratory infection. This transdisciplinary training will facilitate my transition to independence and create a unique research niche on alcohol-induced dysbiosis as a mechanism underlying impaired host defense to bacterial infections. The results from these studies will provide data to support an R01 submission focused on the immunomodulatory effects of alcohol-induced dysbiosis on the gut-lung axis. The mentoring team, the scientific environment, and the research infrastructure provided by the LSUHSC-NO Comprehensive Alcohol-HIV/AIDS Research Center (CARC) will be instrumental in my growth and development as an independent scientist.